Liposomes are artificial vesicular structures composed of single or multiple membranes enclosing an aqueous compartment. Most typically, liposome membranes are formed from lipid bilayers, but they can consist as well from other monomeric and polymeric amphiphilic compounds, including other types of amphiphiles, polymers and polypeptides (Antonietti and Forster 2003). Liposomes form spontaneously when lipids are dispersed in an aqueous environment under suitable conditions. Most liposomes are non-toxic, non-antigenic and biodegradable in character since they have the molecular characteristics of mammalian membranes. Lipophilic or amphiphilic drugs and compounds can be incorporated into the liposome membrane, hydrophilic drugs and compounds can be encapsulated in the aqueous cores of the liposomes.
In recent years liposomes have become an important tool in the pharmaceutical industry for the delivery of drugs (Gregoriadis 1995). Liposomes are capable of influencing pharmacokinetics by a sustained release of the drug to the body or reduce side effects by limiting the free concentration of a drug. By attaching ligands to the liposome or rendering their charge, liposomes facilitate a targeted delivery of drugs to a desired site of action. Beside the pharmaceutical use, liposomes are also frequently used for cosmetic products.
If liposomes are used for the administration of active agents within pharmaceutical or cosmetic use, it is important to control and optimize the loading efficacy of the active compound to the liposomal formulation and the stability of the liposomal formulation loaded with the active or cosmetic compound. The stability of the formulation is a crucial characteristic during manufacturing, storage and application of the formulation. In many cases physical or chemical stability of liposome products is limited, which has to be taken into account for planning of manufacturing processes (hold time), storage (shelf life stability) and application of the product (in-use stability).
For pharmaceutical application liposome formulations are often administrated by injection. Thus the liposomes must be present in an aqueous phase under conditions suitable for intravenous (iv) or intraperitoneal (ip) administration.
Pharmaceutical liposome formulations are subjected to extremely stringent quality criteria. Most present liquid liposome products are not stable over a longer storage period, because they can undergo a variety of chemical and physical degradation processes. However, for pharmaceutical products it is desirable to have final formulations which are stable for at least six months to two years at room temperature or at refrigeration temperature. These factors restrict the use of liposomes as practical carriers of biologically active compounds. For liposomes, techniques for dehydration have been developed to meet these requirements.
Long term stability of liposome formulations is greatly enhanced when they are dehydrated and stored as dry rather than liquid formulations. Before injection, the dry liposome products have to be rehydrated in a suitable aqueous medium generating aqueous suspensions for administration. Because the stability of the rehydrated liposome formulations may again be limited by chemical and physical degradation processes, the increase of the in-use stability of said ready to use liposomal suspension is another important goal of pharmaceutical formulation technology.
A commonly used stabilization method for aqueous liposome suspensions by freeze-drying is described in U.S. Pat. Nos. 4,229,360 and 4,247,411. In the freeze-drying process, a liposomal suspension is frozen and subsequently subjected to reduced pressure, which leads to removal of the frozen water by sublimation. Usually, the aqueous suspension comprises an excipient, such as a sugar, to prevent or minimize defect formation induced by freezing and dehydration. The freeze-drying procedure results in liposomes in a protective matrix of excipient from which, after rehydration, the antecedent liquid product is to be obtained. As disclosed in U.S. Pat. No. 4,880,635 the liposomes can be protected from detrimental effects of the dehydration and rehydration steps by the presence of a protective sugar, not only on the outside, but also inside the liposome.
The spray-drying method, as for example disclosed in U.S. Pat. No. 5,089,181, provides an alternative process for preparing a stable dehydrated liposomal formulation. The process has been adapted from the food industry and employs the atomising of suspensions into small droplets by spraying said suspension, and the subsequent evaporation of the medium from the droplets at elevated temperatures. Like in the freeze drying process, the liposomal suspension may comprise an excipient, such as a sugar, to protect the liposomal membranes. In comparison to freeze-drying, the spray-drying process has considerable advantages with respect to large scale industrial application, because it enables larger manufacturing capacities at lower cost and with manageable technological efforts. However, the elevated temperatures involved in this method apply stress to the encapsulated active agent as well as to the lipid membranes.
To stabilize suspensions which comprise a water soluble drug encapsulated in liposomes for spray drying, U.S. Pat. No. 4,895,719 discloses to balance a high internal osmolarity generated by a high internal concentration of the soluble drug with an evenly high osmolarity of the surrounding medium.
To stabilize hydrophobic drugs in the aqueous phase of liposomes, WO 2007/005754 discloses the complexation of such drugs by cyclodextrins prior to encapsulation. The complexed hydrophobic drug is retained in the liposome at high concentrations, even in the presence of a transmembrane osmotic gradient caused by the cyclodextrin. However a stabilization for active agents embedded in the liposomal membrane is not disclosed.
The presence of solutes that are osmotically active, such as sugars or ions, inside or outside the liposomal membranes generates osmotic forces. The way how transmembrane osmotic gradients act on the structure and dynamics of biological membranes has been investigated in the literature, and models to describe phenomena like stress-strain relation and lysis have been proposed (Ertel, Marangoni et al. 1993; Hallett, Marsh et al. 1993). Briefly, the experiments by the authors and the accompanying analysis underline that swelling of liposomes at a given osmotic stress depends on their size. Swelling up to a size dependent critical yield point was described, at which lysis (leakage) occurred. Conditions under which liposomes are expected to reside in a consistently strained state were given.
With regard to the production of liposomes comprising a lipophilic drug that have an enhanced shelf live, WO 2004/002468 discloses the preparation of liposomes which comprise paclitaxel. The liposomes are formed in an aqueous buffer comprising trehalose, resulting in a liposomal suspension having the same osmolarity inside and outside the liposome. The aqueous liposomal suspension is subsequently dehydrated. The dehydration process may be carried out by freeze-drying or spray-drying. The application discloses several protocols for the freeze drying of said liposomal suspensions. After dehydration, the liposomal preparation can be rehydrated by the addition of water or an aqueous solution. The document does not provide comparative data on the in-use stability of liposomal preparations that were dehydrated by freeze-drying or by spray-drying prior to their rehydration.
In view of the described state of the art, the problem underlying the present invention was the preparation of liposomes, comprising at least one lipohilic active agent and/or cosmetic agent with a high agent to lipid ratio and with improved stability, especially regarding physical stability and release of the agent from the liposome. Especially the invention relates to the problem of providing a process for manufacturing said liposomal preparations that have an extended hold time during manufacturing and in-use stability, wherein the process involves a fast dehydration step.
Thus, the solution of the above problem is achieved according to the present invention by providing embodiments characterized in the claims and further depicted in the description of the invention.